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Metabolic Turnover of Synaptic Proteins: Kinetics Edinburgh Research Explorer Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance Citation for published version: Cohen, LD, Zuchman, R, Sorokina, O, Müller, A, Dieterich, DC, Armstrong, JD, Ziv, T, Ziv, NE & Akaaboune, M (ed.) 2013, 'Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance', PLoS ONE, vol. 8, no. 5, e63191. https://doi.org/10.1371/journal.pone.0063191 Digital Object Identifier (DOI): 10.1371/journal.pone.0063191 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: PLoS ONE General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance Laurie D. Cohen1,2, Rina Zuchman3, Oksana Sorokina4, Anke Mu¨ ller5,6, Daniela C. Dieterich5,6, J. Douglas Armstrong4, Tamar Ziv3, Noam E. Ziv1,2* 1 Technion Faculty of Medicine, Lorry Lokey Center for Life Sciences and Engineering, Technion, Haifa, Israel, 2 Network Biology Research Laboratories, Lorry Lokey Center for Life Sciences and Engineering, Technion, Haifa, Israel, 3 Smoler Proteomics Center, Faculty of Biology, Technion, Haifa, Israel, 4 Institute for Adaptive and Neural Computation, University of Edinburgh, Edinburgh, United Kingdom, 5 Leibniz-Institute for Neurobiology, Magdeburg, Germany, 6 Institute for Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg, Germany Abstract Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (,0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial. Citation: Cohen LD, Zuchman R, Sorokina O, Mu¨ller A, Dieterich DC, et al. (2013) Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance. PLoS ONE 8(5): e63191. doi:10.1371/journal.pone.0063191 Editor: Mohammed Akaaboune, University of Michigan, United States of America Received March 5, 2013; Accepted March 29, 2013; Published May 2, 2013 Copyright: ß 2013 Cohen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has received funding from the United States Israel Binational Science Foundation (2007425), the European Union Seventh Framework Programme under grant agreement nos. HEALTH-F2–2009–241498 (‘‘EUROSPIN’’), and the Deutsch-Israelische-Projektkooperation German-Israeli Project Cooperation foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction remarkably long (e.g. [1,2]), more recent in vitro studies have reported half-lives of synaptic proteins in the range of several hours Chemical synapses contain multitudes of proteins, some of (e.g. [3,4]). Thus, the metabolic cost of maintaining synapses which play direct roles in synaptic transmission, whereas others remains largely unknown. regulate synaptic function or serve as structural scaffolds. Proteins, The elaborate, anisotropic architecture of neurons poses unique including synaptic ones, have finite lifetimes and therefore, need to challenges in terms of synaptic proteostasis: First, synapses, and in be continuously replaced with freshly synthesized copies. Given particular presynaptic compartments, are often located at enor- the huge numbers of synaptic connections each central nervous mous distances from the major site of protein synthesis, namely the system neuron makes, maintenance of synaptic contents would neuronal cell body. Given the enormous lengths axons can attain, conceivably place enormous metabolic demands on individual it might be expected that the life-spans of presynaptic proteins neurons. These demands in turn, depend on anabolic and would generally be longer than those belonging to somatodendritic catabolic rates of synaptic proteins. Surprisingly, perhaps, the compartments. Neurons, however, contain sophisticated and quite turnover kinetics of synaptic proteins have not yet been studied efficient transport mechanisms for delivering particular proteins to systematically. As a result, the estimates for such kinetics vary the far reaches of axons. Yet the transport rates of other synaptic widely. Whereas older studies based on radiolabeling methods proteins can be rather slow – on the order of a few millimeters per indicated that the half-lives of some presynaptic proteins can be day [5–8]. In addition, substantial evidence has accumulated for PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63191 Metabolic Turnover of Synaptic Proteins local synthesis of synaptic proteins in dendrites (reviewed in [9– neuronal cell cultures, during the stage at which most synapto- 12]) and possibly in axons [13,14]. Therefore, relationships genesis has been completed (2–3 weeks in culture), aggressive between turnover rates of particular synaptic proteins and their washes and complete media exchanges are severely detrimental to cellular localization are currently unknown. Moreover, despite neuronal viability. Therefore, rather than replace media, we much evidence for local protein synthesis in dendrites and axons, it added an excess of heavy lysine and arginine. Specifically, after 14 is generally thought that most synaptic proteins, and in particular days in culture, heavy lysine and arginine were added to the presynaptic proteins, are transported from the cell body (e.g. [15]; media, resulting in final concentrations of ,1.9 and ,2.9 mM, but see [16]). It thus remains unclear how the short lifetimes respectively, and final heavy to light (H/L) ratios of ,5:1 for both reported for some synaptic proteins (e.g. [3,4]) are compatible with lysine and arginine. 0, 1, 3, or 7 days later, the neurons were lysed the relatively long times required for trafficking them to their and extracted; the extracts were separated on polyacrylamide gels, remote destinations (reviewed in [16]). which were subsequently cut into 9 sets of bands according to Beyond continual replenishment, protein synthesis is believed to molecular weight. Each gel slice was then subjected to MS play essential roles in driving long-term changes in synaptic analysis, and an H/L ratio for each identified peptide was composition and function. Moreover, local synthesis and degra- determined. H/L ratios for all peptides belonging to a particular dation processes have been suggested to affect the properties of protein species were pooled, providing an average H/L ratio for specific synapses by changing the abundance of particular synaptic each protein. The entire process is illustrated in Fig. 1. molecules in a spatially confined
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